This invention relates to a method and an apparatus for disrupting cells in a fluid suspension by means of a continuous process.
The disruption of cells is an important step in many biotechnological processes. Although some biological products are secreted by the cells or released by autolysis, many others, including vaccines, therapeutic substances, enzymatic and diagnostic preparations, must be obtained by disintegrating the cells in order to isolate the product molecules or other subcellular components, such as the membrane.
In the laboratory, cells are usually disintegrated by means of mechanical, physical (ultrasound), chemical and biological processes.
At industrial level, on the other hand, high-pressure technology or bead mills are generally used. In some special cases enzymatic and chemical processes may be used.
All areas of biotechnology, especially those that make use of recombinant and pathogenic microorganisms or their cellular components, can benefit from the use of controlled cell disruption processes that do not involve any biological hazards (containment) and can be certified. Biotechnological processes must be designed and implemented to comply with the applicable process containment and decontamination safety standards. If possible, all equipment in any way associated with the process should be certified.
Very often the aim of a cell disruption process is to achieve productive and limited disruption, but the choice of equipment for the downstream process is dictated by the need to comply with specific containment and hazard prevention requirements when processing certain categories of cells such as genetically modified microorganisms (OGMs) and pathogens. Up until now the use of protein-secreting microorganisms and the subsequent separation of the product by filtering or centrifugation have influenced the use of cell disrupting equipment, considered inadequate in terms of containment, although there have been some recent developments in this area.
Cell disruption is usually assessed subjectively and empirically by inspecting the cell broth (colour, optical density, product viscosity). However, in order to perform an objective assessment the product must be analysed by measuring the size of the particles before and after the process and observing their physical integrity, or by measuring the extracellular activity of an indicator enzyme. Microscopy, preferably using a phase contrast optical microscope that is also capable of recognizing any partially disrupted cells, is a fast and reliable method for assessing the level of cell disruption. This is extremely important for the downstream process, as the product can be released even in the event of partial disruption, while the remaining particle is big enough to facilitate the centrifugal separation process. The most suitable method for use in a production process still consists of analyzing the product directly or measuring its activity.
The ideal cell disruptor should satisfy all of the following criteria: be capable of disrupting even the hardest microorganisms without destroying the intracellular material; be controllable and reproducible; have CIP and SIP capabilities; be compatible with the implementation of biohazard control procedures (containment); ensure compliance with the applicable pharmaceutical standards; be capable of ensuring disruption with a single passage in a continuous process in order to prevent denaturation and reduce processing times and costs; have controlled heat generation (to prevent denaturation); be automation-compatible; be capable of processing volumes that are consistent with the plant's fermentation/separation capacity; be capable of continuous operation; have low operating costs (low energy consumption, require only occasional maintenance, spare parts must be cheap and readily available); require a limited initial outlay; be compact.
As regards controlled heat generation, in an ideal cell disruptor overheating should be avoided by means of adequate cooling before and after disruption, using a heat exchanger.
There are various methods for performing cell disruption.
A first method consists of disruption by means of thermal shock, or hot/cold treatment. This widely used method is also the most traditional; it is also simple and not particularly expensive. Since this method is absolutely non-selective, a possible secondary effect could be the denaturation of the intracellular substances.
A second method consists of disrupting the microbial cells biologically. Much research has been carried out into the action of enzymes and chemical substances and we have adequate information as regards the formation or dissociation of specific bonds and the concurrent loss of integrity of the structural macromolecules in the cell wall or membrane, resulting in the lysis of the bonds that form the membrane or cell wall. This method is highly selective and precise but preparation is complex and costly and it is not suitable for scale-up.
A third method consists of disruption using chemical substances. Detergents, solvents and acids are usually added to the cell broths to induce the death of the cells and subsequent disruption. This method is sufficiently specific and not particularly expensive, but has repercussions on the end product: the substances that are added contaminate the end product and must be removed and eliminated.
A fourth method is based on the use of ultrasound technology, or sonication. This method is only suitable for laboratory use and generates a great deal of heat that is transferred to the processed product.
A fifth method, which is not as well known and is less commonly used, consists of mechanical cell disruption.
Some mechanical systems, such as bead mills, use shearing forces to break the cells. This is a reliable and reproducible method; however continuous operation is not possible, processing is slow and the equipment is not easy to clean.
A sixth method concerns the use of high-pressure mechanical systems. Cell disruption is induced by the sudden passage from a high-pressure zone to a low-pressure zone, with or without impaction, which causes the cells to break.
There are two types of high-pressure mechanical systems: those that use isostatic pressure and those that use dynamic pressure. Isostatic pressure is used in isostatic presses. These machines are extremely efficient but very expensive in terms of the initial outlay and also as far as energy consumption is concerned. The process is discontinuous and is not easily adapted to suit different production requirements, given the small volumes involved.
High-pressure homogenizers use dynamic pressure. These systems are highly reproducible and also available on a large scale. They are extremely easy to use and are suitable for CIP and SIP cleaning procedures.
The dynamic high-pressure system best satisfies the criteria listed above for the ideal cell disruption method, especially for liquid products.
In the prior art the maximum pressure that can be applied is 1500 bar, both on a laboratory and industrial scale. This enables good results to be achieved but requires several passages through the machine (recirculation).
U.S. Pat. No. 4,773,833 describes a homogenizer comprising a homogenizing valve mounted on a pump assembly. The pump has a single pump head, but comprises an intake duct with a hemispherical end part and a delivery duct with a hemispherical end part that lead into a hemispherical chamber in the pump, thus eliminating all the sharp corners and giving the inside of the head a specific shape to improve fatigue strength. However, this type of configuration does not easily withstand pressures of above 1000 bar.
Patent PR99A000045 by the author of this patent application relates to a high-pressure fluid pump comprising a floating plunger in a pumping chamber in which the fluid is pumped from a fluid intake zone to a fluid delivery zone; a block for each piston, to connect the pumping chamber to the intake and delivery valves housed in containers to the side that are fastened to the block. Each block comprises two semi-parts or plates that are clamped together and have grooves on the inside that house an internal manifold connecting the pumping chamber with the intake and delivery valves.